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Normal segregation of a foreign-species chromosome during Drosophila

female meiosis despite extensive heterochromatin divergence

William D. Gillilanda,1, Eileen M. Colwella, David M. Osieckia, Suna

Parkb, Deanna Linb, Chandramouli Rathnamb, and Daniel A. Barbashb,1 a Department of Biological Sciences, DePaul University, Chicago, IL 60614 b Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853 1 To whom correspondence should be addressed: Email: wgillila@depaul.edu or barbash@cornell.edu

Genetics: Early Online, published on November 17, 2014 as 10.1534/genetics.114.172072

Copyright 2014.

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Abstract The abundance and composition of heterochromatin changes rapidly between

species and contributes to hybrid incompatibility and reproductive isolation.

Heterochromatin differences may also destabilize chromosome segregation and

cause meiotic drive, the non-Mendelian segregation of homologous chromosomes.

Here we use a range of genetic and cytological assays to examine the meiotic

properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D.

melanogaster. These two species differ by ~12-13% at synonymous sites and

several genes essential for chromosome segregation have experienced recurrent

adaptive evolution since their divergence. Furthermore, their chromosome 4s are

visibly different due to heterochromatin divergence, including in the AATAT

pericentromeric satellite DNA. We find a visible imbalance in the positioning of the

two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding

with a D. melanogaster 4 containing a heterochromatic deletion. These results

demonstrate that heterochromatin abundance can have a visible effect on

chromosome positioning during meiosis. Despite this effect, however, we find that

sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females, and

does not experience elevated non-disjunction. We conclude that segregation

abnormalities and a high level of meiotic drive are not inevitable byproducts of

extensive heterochromatin divergence.

Article Summary Animal chromosomes typically contain large amounts of non-coding repetitive

DNA that nevertheless varies widely between species. This variation may potentially

induce non-Mendelian transmission of chromosomes. We have examined the

meiotic properties and transmission of a highly diverged chromosome 4 from a

foreign species within the fruitfly Drosophila melanogaster. This chromosome has

substantially less of a simple sequence repeat than does D. melanogaster 4, and we

find that this difference results in altered positioning when chromosomes align during

meiosis. Yet this foreign chromosome segregates at normal frequencies,

demonstrating that chromosome segregation can be robust to major differences in

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repetitive DNA abundance.

Introduction

Heterochromatic repeats at and near telomeres and centromeres turnover

rapidly at short evolutionary time scales (Charlesworth et al., 1994). A subset of

genes involved in meiosis, chromosome and chromatin function, and transposable

element defense also show high rates of divergence between sibling species, often

with accompanying signatures of adaptive evolution (Anderson et al., 2009; Begun

et al., 2007; Langley et al., 2012; Larracuente et al., 2008; Malik and Henikoff, 2001;

Obbard et al., 2009; Raffa et al., 2011). These patterns suggest that organisms

need to mount a continual adaptive response to suppress deleterious consequences

caused by heterochromatic repetitive DNAs. Satellite DNAs and transposable

elements, the major components of heterochromatin, can increase their copy

numbers by unequal crossing over and transposition. These expansions can reduce

fitness by increasing genome size and rates of ectopic recombination.

Repetitive DNA evolution can be particularly rapid if it selfishly biases its

transmission through meiosis (true meiotic drive) or gametogenesis (gametic drive;

we refer to both phenomena collectively as segregation distortion). Meiotic drive is

an especially strong driver of chromosomal evolution that takes advantage of

asymmetric meioses (that is, females in Drosophila and mammals) where only one

meiotic product becomes the egg pronucleus (Fabritius et al., 2011; Pardo-Manuel

de Villena and Sapienza, 2001). The selfish elements that cause meiotic drive likely

result from variation in heterochromatic repeat sequences (Buckler et al., 1999;

Fishman and Saunders, 2008). Adaptive divergence of centromeric and telomeric

proteins may reflect a host response to suppress meiotic drive, as meiotic drivers

can have pleiotropic deleterious consequences on host fitness (Henikoff et al., 2001;

Zwick et al., 1999).

There are hints that segregation distorters may be prevalent in natural

populations (Bastide et al., 2013; Jaenike, 2001; Reed et al., 2005), but few specific

loci have been identified. Hybrid backgrounds may reveal these loci, if suppressors

fail to function or are separated from their targets by segregation (Mercot et al.,

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1995). Here we take advantage of a rare opportunity to examine meiotic

transmission of an entire foreign chromosome, that is Drosophila simulans

chromosome 4 (sim-IV) in a heterospecific D. melanogaster background. D.

melanogaster and D. simulans are sibling species that can be intercrossed but

contain substantial divergence. Alignable synonymous nucleotide sites are ~12-13%

diverged (Begun et al., 2007), and the species are strikingly different in repetitive

DNA content and heterochromatin, with D. simulans having substantially less

transposable elements and satellite DNA (Bosco et al., 2007; Lerat et al., 2011;

Lohe and Roberts, 1988). They also have experienced adaptive evolution in genes

that are essential for chromosome segregation (Anderson et al., 2009; Malik and

Henikoff, 2001).

Chromosome 4 has a number of advantages for this study. 1) sim-IV is viable

when introgressed into D. melanogaster due to its small size, the only incompatible

phenotype being homozygous male sterility (Muller and Pontecorvo, 1942). 2)

Chromosome 4 is triplo-viable, which allows for novel chromosome segregation

assays (Sturtevant, 1934). 3) Chromosome 4 contains an interesting mix of

heterochromatic and euchromatic properties (Riddle et al., 2009). It has a high

proportion of repetitive DNA but a normal abundance of protein coding genes. It is

therefore not a gene-poor B or Y chromosome. 4) Chromosome 4 is achiasmatic

and segregates in the absence of crossing over. Therefore all divergence on 4

remains linked to the centromere and can potentially impact meiotic segregation. 5)

Chromosome 4 segregation nevertheless typically utilizes homology to achieve

pairing during meiosis, while also being able to segregate under an alternative

homology-independent pathway when homology is absent (Hawley et al., 1992). In

short, we propose that we are testing for faithful segregation among the most

diverged chromosomes possible in an animal model.

One recent advance in understanding the segregation of nonexchange

chromosomes, such as the small 4 chromosomes of Drosophila, is the identification

of tethers connecting spatially separated chromosomes during prometaphase of

meiosis I in females. These tethers appear to be built from pericentromeric

heterochromatin and are proposed to establish tension between chromosomes not

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held together by chiasmata, thus allowing homologous coorientation to be

established (Hughes et al., 2009; Hughes et al., 2011). Similar tethers have been

inferred by micromanipulation experiments in grasshopper spermatocytes

(LaFountain et al., 2002) and by PICH localization to DNA threads connecting mitotic

sister kinetochores in mammalian cultured cells (Baumann et al., 2007). While the

exact mechanisms of establishing and resolving these tethers are unknown, they are

a strong candidate for establishing nonexchange chromosome segregation, as

heterochromatic homology is sufficient for coorientation (Hawley et al., 1992).

Heterochromatin divergence between species can cause mitotic segregation failure

in interspecific hybrids (Ferree and Barbash, 2009). Here we address whether a

foreign-species chromosome with extensive divergence affects the formation of

heterochromatic threads and can segregate properly during female meiosis.

Results

Reduced heterochromatin of sim-IV. In examining sim-IV, in comparison with

pure-strain D. melanogaster and D. simulans oocytes, we found that sim-IV is

dimmer than its D. melanogaster homolog in DAPI fluorescence. This was readily

apparent even in the ocular, and caused an asymmetry between the 4s in

heterozygous females (Fig. 1A-C). This dimness, without asymmetry, was also

observed in introgressed sim-IV homozygotes (Fig. 1D) as well as D. simulans

females (Fig. 1E). This result is not unexpected; the AATAT heterochromatin repeat,

which primarily labels the 4 in females (Dernburg, 2000), is considerably less

abundant in the D. simulans genome, comprising only 1.9% of the genome versus

3.1% in D. melanogaster (Lohe and Brutlag, 1987).

Positioning of sim-IV during female meiosis. Because recent work has

identified heterochromatin tethers that can incorporate the AATAT repeat (Hughes et

al., 2009), we asked whether these tethers were also present in D. simulans. We

were able to detect them by both a phospho-specific histone antibody that can

highlight threads (Hughes et al., 2011) and by fluorescent in situ hybridization (FISH)

of an AATAT probe (Fig. 2). However, during this experiment, we noticed that it was

much more difficult to find oocytes that had their chromosome 4s positioned far

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enough out on the spindle to have detectable threads, in both D. simulans and

introgressed sim-IV females. Instead, while roughly similar numbers of oocytes

appeared to have chromosomes out on the spindle (and therefore also roughly equal

durations of time spent in prometaphase), those chromosomes were positioned

much closer to the main mass of chiasmate chromosomes. To quantify this, we did

preps under tightly controlled aging and dissection conditions, and measured the 4-4

distances for oocytes from pure-strain D. melanogaster, introgressed sim-IV hetero-

and homozygotes, and pure-strain D. simulans (Fig. 3). To limit consideration to 4-4

distances under comparable conditions of prometaphase and congression, we

excluded those oocytes where other chromosomes besides the 4 were

spontaneously nonexchange, as well as oocytes that were fixed while chromosomes

were in transient configurations such as having both homologs on the same side of

the spindle (Hughes et al., 2009) or in the “slippage” configuration where the

chiasmate autosomes are positioned end-to-end (Hughes et al., 2011).

Consistent with our initial qualitative observations, we found that the mean 4-4

distances in pure-strain (pol/pol) D. melanogaster females (11.3 µm) were nearly

twice as large as in D. simulans (6.1 µm). Interestingly, the introgressed sim-IV

chromosome was more intermediate when homozygous in D. melanogaster (sim-

IV/sim-IV: 8.1 µm), suggesting that genetic background affects chromosome

positioning. This may also contribute to the difference between the two

heterozygous genotypes (sim-IV/pol: 8.7 µm, sim-IV/ciD: 6.79 µm). Note that

because the 4 chromosomes are normally positioned near the centromeres of the

other chromosomes, the minimum 4 separation is the normal karyosome width,

approximately 4.5 µm. Therefore the proportional separation of 4 chromosomes from

the main mass is considerably larger in pure-strain D. melanogaster. Many of these

comparisons, including all comparisons involving pure-strain D. melanogaster, were

highly statistically significant as determined by pairwise t-tests (Fig. S1).

This novel observation that the 4th chromosomes from these two closely

related species have notably different behavior provides strong evidence that the

amount of heterochromatin on a chromosome has a functional consequence. A

speculative further interpretation is that if the repeats on a chromosome are forming

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threads that connect nonexchange homologs, then having a greater amount of those

repeats may increase thread length and enable those homologs to move farther

apart from each other before the tether pulls tight enough to prevent further

movement.

Reducing AATAT content also affects positioning of D. melanogaster 4. This simple model suggests that deleting some of the 4 heterochromatin should

reduce the 4-4 distance during prometaphase. Few deletions on the D.

melanogaster 4 chromosome are available, but Df(4)M101-62f deletes proximal

gene-containing sequence and extends into the centromeric heterochromatin for an

unknown distance (John Locke, Pers. Comm.). We crossed this deletion to the same

pol stock used above to produce Df(4)m101-62f/pol females. We found that the

deficiency chromosome was noticeably smaller than pol, and hybridized less

strongly to the AATAT FISH probe (Figure 4), consistent with the deletion of some of

the 4 heterochromatin. Then, we measured the 4-4 distances in oocytes from

Df(4)m101-62f/pol females, and found a highly significant reduction in the mean 4-4

distance (6.8 µm, Fig. 3, Fig. S1). These results strongly support our conclusion that

4-4 distances are proportional to the amount of 4 heterochromatin.

Segregation of sim-IV in D. melanogaster females. To test whether sim-IV

segregates properly in a foreign species, we assayed sim-IV by making it

heterozygous over a y+-marked D. melanogaster reference chromosome in D.

melanogaster females. We also performed in parallel a control cross using a w+-

marked D. melanogaster chromosome 4 that was heterozygous over the same

reference chromosome (Figure 5). Over 4400 progeny were scored in each

experiment (Table 1). In the control cross the two progeny classes were not

significantly different from the expected 1:1 ratio. In the experimental cross sim-IV

progeny were recovered at slightly below Mendelian expectations (47.8%). This

deficit, however, is significantly below 50% (p<0.002, binomial simulation). The

experiment and control are also significantly different when compared directly in a

contingency table (p<0.05, Chi Square).

Normal disjunction of sim-IV in D. melanogaster. These differences might

reflect a true segregation disadvantage of sim-IV, but also could result from small

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viability differences between D. melanogaster flies heterozygous for sim-IV versus

mel-IV that cannot be easily detected. We therefore performed a range of additional

assays. First we measured non-disjunction (NDJ) within the above cross, since it

can result from chromosome loss, the most plausible cause of reduced transmission.

The absolute rate in sim-IV/y+ females was 2.3 x 10-4, lower than in the

corresponding control and consistent with wild type rates for pure-strain D.

melanogaster from other published studies (see Figure 5).

We further tested the meiotic behavior of sim-IV by crossing to males from a

standard NDJ tester stock that allows estimation of both X and 4 NDJ. We observed

no X or 4 NDJ within the sim-IV introgression stock, either as sim-IV/ciD

heterozygotes or sim-IV/sim-IV homozygotes (Table 2). We also outcrossed the

stock to a standard laboratory stock with the 4th chromosome marked with pol, to

create sim-IV/pol females, and again saw no X or 4 NDJ in this genotype. Because

of these negative results, we considered the possibility that any defect in sim-IV may

be weak. We reasoned that if this were the case, we might see NDJ if we sensitized

the genetic background to increase NDJ, as has been done for assaying natural

variation (Zwick et al., 1999). We performed two sensitizations, one by testing sim-IV

in a background carrying a single dose of the meiotic mutant nod, and the other by

testing sim-IV in females heterozygous for the X chromosome balancer FM7. Even

in these sensitized backgrounds, we saw no increase in NDJ (Table 2). Furthermore,

the transmission rates appear roughly equal for both 4th chromosomes, by

comparing the pol- minute and pol+ minute progeny of heterozygous sim-IV/pol

females. Therefore, the genetic evidence from a range of genetic backgrounds

strongly suggests that the introgressed sim-IV chromosome is fully competent for

normal segregation in female meiosis.

Normal sim-IV segregation in triplo-4 D. melanogaster females. Females

carrying three chromosome 4s are viable and fertile. Such females are expected to

produce three types of meiotic segregation at equal frequencies (Figure 6).

Sturtevant discovered, however, that in many crosses with triplo 4s the segregation

ratios differ substantially from equal frequencies (Sturtevant, 1934; Sturtevant,

1936). He further determined that different chromosome 4s from wild type and

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marker strains display a characteristic “preference” for whether they tend to

segregate with one of the other chromosome 4s being tested (classes I and III in

Figure 6), or instead segregate away from the other 2 chromosome 4s (class II).

The genetic basis of this curious preference property remains unexplained. In our

scheme we arranged in a triplo-4 female the unmarked chromosome to be tested

against chromosome 4s dominantly marked with either y+ or w+ (Figure 6). We

reasoned that if sim-IV is perceived by D. melanogaster as being a foreign

chromosome, then the 2 marked D. melanogaster 4s would segregate away from

each other and sim-IV would segregate analogous to a free duplication. This would

result in a deficit of type II segregation below the random expectation of 1/3, which

would manifest as a deficit of y+ w+ and y w phenotypes.

Contrary to this expectation we found that class II segregations were

significantly over-represented with sim-IV, but also in 4 out of 5 control crosses with

D. melanogaster chromosome 4s derived from different marker and wild type stocks

(Table 3). The one outlier with a significant deficit of class II segregations involved

chromosome 4 from the wild type stock BS 1. The wide range of values is

consistent with results from Sturtevant (Sturtevant, 1936). This variation is not due to

aberrant production or recovery of the 2 reciprocal classes within the 3 segregation

types, because in most crosses the number of y+ progeny was similar to w+ progeny

produced by class I and III segregations, and likewise for y+ w+ and y w progeny

produced by class II segregation. Instead we conclude that sim-IV segregation falls

with the normal range of variation observed for D. melanogaster chromosome 4s.

Discussion

The function of heterochromatic threads in meiosis. The heterochromatic

threads connecting homologous chromosomes in female meiosis are the leading

candidate mechanism for how nonexchange chromosomes achieve proper

coorientation (Hughes et al., 2009), as they can explain a variety of experimental

observations, such as heterochromatic homology being sufficient to achieve

segregation (Hawley et al., 1992). We found that sim-IV has shortened threads and

is positioned more closely to the other chromosomes compared to mel-IV. We

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suggest that this correlation reflects a role of threads in chromosome positioning, but

acknowledge that differential positioning might have other causes such as variation

in microtubule capture or centromere strength. Regardless, we have also found that

both properties correlate with differences in heterochromatin abundance, both

between mel-IV and sim-IV, and between wild type mel-IV and a heterochromatic

deletion. Our results therefore provide evidence that the amount of heterochromatin

on the 4 changes its positioning.

In addition to unresolved questions of the proximal mechanism (such as how

threads are established, how they regulate coorientation, and how they are finally

resolved), there is also the evolutionary question of why these chromosomes move

out on the spindle at all. We suggest that because chromosome 4 is fully

achiasmatic, it may be acting as an “organizing center” for threads emanating from

other chromosomes. This idea is conceptually similar to a proposal by A.T.C.

Carpenter (Carpenter, 1991), with chromatin threads fulfilling the role previously

proposed for interchromosomal microtubules. There is some circumstantial evidence

for this organizational role; for example, the microtubule mass along the spindle arc

between prometaphase 4 chromosomes is substantially denser than elsewhere in

the spindle (Hawley and Theurkauf, 1993) and in some figures threads that appear

to originate from other chromosomes can also lead towards the 4s (Hughes et al.,

2009). We further suggest that increased amounts of heterochromatin on 4 cause

longer threads. These longer threads may more efficiently capture or associate with

heterochromatic threads from facultatively achiasmate chromosomes and increase

their probability of correct segregation.

If so, this role suggests parallels between the evolution of heterochromatin and

other aspects of meiosis. While D. melanogaster has many common polymorphic

chromosome inversions, D. simulans is monomorphic with no common inversions

(Lemeunier and Aulard, 1992). As inversions block crossing over, increasing the

abundance of inversions will make meioses with nonexchange chromosomes more

common. In D. melanogaster, nonexchange chromosomes move out on the spindle

during prometaphase I. While the significance of this movement is not known, we

speculate that it may be involved in how the oocyte achieves proper nonexchange

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chromosome coorientation and metaphase-arrested karyosome structure. Because

nonexchange chromosomes in D. melanogaster are positioned between the 4s near

the spindle poles and the exchange chromosomes at the metaphase plate, having

the 4s further out would provide more space for additional nonexchange

chromosomes to also move fully out onto the spindle. If this additional space is

beneficial (such as reducing the time needed to complete prometaphase, or avoiding

deleterious entanglements between multiple nonexchange chromosomes), then the

greater amount of space on the spindle provided by the longer 4-4 tethers in D.

melanogaster may help this species to tolerate common inversions. Note that the

causal relationship in this model is unknown; it could be that longer 4-4 tethers

evolved first, which allowed inversions to accumulate in the population, or

alternatively, accumulating inversions favored the evolution of longer tethers to

accommodate their segregation. Either way, this model predicts that Drosophila

species with common inversions should have greater 4-4 distances than species

that lack them. This would be particularly interesting to examine in species such as

D. virilis, which has a large genome with a high satellite DNA content (Bosco et al.,

2007), yet appears to lack inversions in natural populations (Evgen'ev et al., 2000).

This hypothesis also may explain why dot chromosomes persist in many Drosophila

species (Ashburner et al., 2005).

Heterochromatin divergence and meiotic drive. There is a resurgence of

interest in heterochromatin variation, due to evidence that it affects gene expression

(Lemos et al., 2010) and to new methods to detect and quantitate such variation

(Aldrich and Maggert, 2014). Strong meiotic drive is typically associated with

cytologically detectable differences in heterochromatin between chromosomes

(Dawe, 2009; Fishman and Saunders, 2008). Our results here show that a large

difference in abundance of the AATAT satellite between D. simulans and D.

melanogaster chromosome 4s does not result in similarly dramatic levels of meiotic

drive. We suggest that location as well as abundance influences whether satellite

DNA blocks affect centromere behavior or take on neo-centromere function,

analogous to heterochromatin position effects that are proposed to influence

whether or not circularized sex chromosomes cause mitotic defects (Ferree et al.,

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2014). Our results further suggest that strong meiotic drive is not an inevitable

consequence of even extensive chromosome divergence. It remains an open

question whether meiotic drivers are truly rare in nature, or instead whether higher

frequency variants exist that cause lower level drive that is beyond the limit of

detection in small-scale experiments. A major hurdle in resolving this question is the

difficulty of reliably detecting weak meiotic drive effects, one example being the

maize chromosomal knob K10L2 (Kanizay et al., 2013).

Faithful segregation of sim-IV. Our diplo segregation assay did reveal a

small (~2%) but statistically significant deficit in sim-IV-containing progeny. However

this deficit is well within the range of potential viability effects. Distinguishing subtle

viability effects versus a meiotic segregation difference would require precise

tracking and quantification of egg to adult viability for many thousands of animals.

We instead pursued two additional approaches to examine sim-IV segregation. First

we quantitated non-disjunction in a manner that includes the detection of

chromosome loss events. We found no excess in NDJ for sim-IV, most strikingly

even when sensitizing the genetic background using either a nod mutation or an

achiasmate X chromosome balancer.

Segregation of sim-IV in triplo-4 females. Our second approach took

advantage of the very high levels of non-random disjunction that are often seen in

triplo 4 females. We constructed D. melanogaster females containing sim-IV as the

tester chromosome and two marked D. melanogaster 4s, as well as 5 control lines

with different tester D. melanogaster 4s. We expected that if sim-IV is “perceived”

as being foreign or distinct from D. melanogaster 4s, then the two D. melanogaster

4s would preferentially segregate away from each other, resulting in an excess of

class I and III segregations and a deficit of class II (Table 3). Instead we saw the

opposite pattern, with 45.9% class II segregations compared to the random

expectation of 33.3%.

It is instructive to compare this result to cases where chromosome 4 derivatives

or aberrations have been introduced into diplo 4 backgrounds, even if the use of

different reference 4s between studies precludes precise quantitative comparisons.

Hawley et al. (Table 3 in ref. Hawley et al., 1992) examined the effects of a series of

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Dp(1;4) chromosomes containing varying amounts of chromosome 4

heterochromatin on segregation of two marked chromosome 4s. NDJ of these two

4s is analogous to class II segregation in Fig. 6. NDJ ranged from ~12-33% and

showed a positive correlation with abundance of chromosome 4 heterochromatin.

Interestingly, a deletion derivative, Dp(1;4)M5D, that appears to remove some

chromosome 4 heterochromatin induced very low NDJ. Similarly, Bauerly et al.

recently discovered D. melanogaster strains containing B chromosomes that are

predominantly composed of AATAT satellite and may be derived from chromosome

4s (Bauerly et al., 2014). These B chromosomes induced 27.1% chromosome 4

NDJ. These results make all the more striking the fact that sim-IV induces a very

high frequency of class II segregations despite having reduced AATAT content.

Materials and Methods

Drosophila stocks and nomenclature. We refer to generic fourth

chromosomes as 4, and specific fourth chromosomes as IV. Therefore, the

unmarked introgressed D. simulans 4th chromosome used in this study is referred to

as sim-IV. An exception is the D. melanogaster chromosome 4 containing the visible

eye marker svspa-pol, which we refer to simply as pol. The 4 wild type lines used in

triplo-4 segregation assay were obtained from Dr. Stuart MacDonald and are

described elsewhere (King et al., 2012). We created a D. melanogaster y w sim-

IV/ciD stock derived from the sim-IV introgression obtained from Dr. JP Masly (Masly

et al., 2006). All other stocks were from the Hawley lab or obtained from the

Bloomington Drosophila Stock Center. We used a w+-marked chromosome 4 (y1

w1118; PBac{w+mC=5HPw+}CG33978A437), abbreviated as w+-IV as a control

chromosome in crosses in Table 1 to measure sim-IV segregation and production of

nullo maternal gametes. A y+-marked chromosome 4 (y1 w1118; PBac{y+-attP-

9A}VK00024), abbreviated as y+-IV, was used as the opposing chromosome to

follow segregation of the sim-IV or control chromosome.

Drosophila crosses. In the C(4)RM, ci1 eyR stock used in Table 1 the

penetrance of the ey phenotype was variable. Among the thousands of progeny a

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small number of various developmental defects were observed. Therefore flies were

scored as being ci ey only if both wings displayed the ci1 phenotype and at least one

eye displayed a small or misshapen eye characteristic of the eyR phenotype. In the

experimental cross ci ey females will be y w+, and ci ey males will be y w. Regular

progeny with these phenotypes are thus potentially overlapping with C(4)/O if the

regular progeny have morphological defects affecting the wings and eyes. Between

2 and 11 flies with morphological defects were found for each sex and genotype in

the Table 1 crosses, and were predominantly cases where one eye was missing and

wings were wild type or where both eyes were wild type and one wing had a

defective longitudinal vein 4 or 5. In the control cross ci ey females will be y w+, and

ci ey males will be y w. No regular y w males will be produced but regular y w+

daughters are again potentially overlapping with C(4)/O. We also found the minute

phenotype associated with haplo-4 challenging to score but classified between 2 and

17 flies of each sex and genotype as minute in Table 1.

To measure NDJ in the y w; sim-IV/sim-IV, y w; sim-IV/pol and y w; sim-IV/ciD

genotypes, single virgin females were mated to multiple C(1;Y), v f B/O; C(4)RM, ci

eyR/O males in vials, allowed to lay eggs for 5 days, and adults removed. X

chromosome NDJ could be seen by following y (normal progeny were y+ females

and y– males, while progeny of diplo-X or nullo-X eggs were y– females and y+

males, respectively). Progeny of nullo-4 eggs could be identified as being both ci

and ey (normal progeny in the sim-IV/ciD cross could be ci alone), but because the

sim-IV chromosome is wildtype for all chromosome 4 markers, diplo-4 progeny of

mothers carrying sim-IV could not be distinguished from normal progeny.

To produce y w; sim-IV/pol females, we crossed y w; sim-IV homozygous

females from the introgression stock to males from a y w/y+Y; pol laboratory stock.

Then y w / y+Y; pol/sim-IV heterozgous males were collected and backcrossed to

y w; pol virgin females to produce y w; pol/sim-IV females.

To produce FM7, y w B / y w; pol/sim-IV and FM7, y w B / y w; sim-IV/sim-IV

females, y w / y+Y; sim-IV/pol males from above were crossed to FM7, y w B; pol

females, and FM7, y w B/y+Y; sim-IV/pol males and FM7, y w B/y w; sim-IV/pol virgin

females were collected. These were sib-mated, which produced FM7, y w B / y w

  15  

females that were phenotypically pol+. These females could be either pol/sim-IV or

sim-IV/sim-IV, which were expected in a 2:1 ratio. These females were mated singly

in vials to C(1;Y)/O; C(4)/O tester males to test X and 4 NDJ as above. The maternal

4 genotype was inferred to be sim-IV/pol if any pol minute progeny were produced in

a vial. Vials that did not produce any pol minute progeny were also testcrossed by

mating multiple F2 females to y w/y+Y; pol males and looking for any pol progeny; all

tested vials were confirmed to lack pol meaning the experimental female in that vial

must have been sim-IV/sim-IV. Count data for each vial were then combined by

maternal 4 genotype.

To produce y w / y w noda; pol and y w / y w noda; sim-IV/pol progeny, y w noda

/ y+Y; pol males (from a stock with the X balanced over C(1)DX females) were

crossed to FM7, y w B / y w; pol/sim-IV virgin females from above, and virgin

females of both genotypes were collected and mated singly in vials to C(1;Y)/O;

C(4)/O tester males as above.

To produce triplo-4 females, we used a mutation in nod to increase the rate of

non-disjunction. The w+-IV chromosome was crossed into a FM7a, nod background

to generate the stock C(1)DX, y1 w1 f1/ FM7a, nod4/ /Dp(1;Y)y+;

PBac{w+mC=5HPw+}CG33978A437. We abbreviate the males from this stock as

FM7a, nod4/Y; w+-IV. To generate triplo-4 females, we first crossed y w; y+-IV

females to FM7a, nod4/Y; w+-IV males. F1 virgin daughters of genotype y w/ FM7a,

nod4/Y; y+-IV/w+-IV were then mated to males of genotype y w/Y containing different

chromosome 4 genotypes. Males containing wild type chromosome 4s were

generated by crossing y w; sim-IV/ciD females to wild type males, and selecting y

w/Y; +/ciD sons. Rare y w/y w daughters inheriting both maternal chromosome 4s

and a paternal chromosome 4 were identified by their y+ w+ phenotype; where

appropriate non-ciD females were selected in order to obtain the desired paternally

inherited wild type chromosome 4. Triplo-4 females were then mated singly to 2 y

w/Y males at 25°.

Probability analyses were done in R (cran.r-project.org). To test significance for

random segregation with 50% survival in Table 1, a binomial number Nj was

generated with a mean of 0.5 and an N of twice the experimental result. The

  16  

surviving segregation proportion was then simulated as pj = binomial(0.5, Nj) / Nj,

This was repeated 1,000,000 times to generate a distribution, with significance

determined as the two-tailed likelihood of obtaining the observed result due to

chance.

4-4 Distance Preps. Bottles were cleared of adults and virgin females of the

desired genotypes were collected 6 hours later. Females were aged in yeasted vials

with sibling males for 42 hours after collection, and so were 42-48 hours post

eclosion at the point of dissection. To standardize prep conditions, a timer was

started as the vial was anesthetized with CO2, followed by hand-dissection of

ovaries as quickly as possible in room temperature 1x Robb’s media + 1% BSA

(Matthies et al., 2000), transferring ovaries to a second well of media after

extraction. After ten females were dissected, the ovaries were left to incubate in

Robb’s until the timer reached 7 min, when buffer plus ovaries were pipetted into a

1.5 mL eppendorf tube and allowed to settle. At 8 min the Robb’s was aspirated, and

1.3 mL of room temperature fixative (a 1:1 mix of 16% EM grade paraformaldehyde

(Ted Pella) with William’s Hypotonic Oocyte Preservation and Stabilization Solution

(Gillies et al., 2013), combined just before use) was applied. After fixation at room

temperature for 5 min, oocytes were washed briefly in PBST (PBS + 0.1% Triton-X

100), ovarioles were separated by rapid pipetting with a p1000 pipette, washed 3x in

PBST for 15 min each, stained in PBST plus 1x DAPI for 6 min, washed in PBST (3x

quickly followed by 2x 15 min) then mounted on slides in SlowFade Gold

(Invitrogen).

Fluorescent in-situ Hybridization (FISH) Preps. Females were aged for 2 or

3 days post eclosion in yeasted vials with males. A timer was started as females

were anesthetized with CO2, transferred to a CO2 plate for 1 min, then the gas was

turned off, flies were covered with a petri dish lid and allowed to rest on the plate. At

6 min, the CO2 was turned back on, and ovaries were dissected as quickly as

possible in Robb’s (above). Once all ovaries were dissected, they were left to

incubate in Robb’s until 15 min from the start of the procedure, when they were

  17  

transferred to an eppendorf tube. Oocytes were allowed to settle for one min, the

Robb’s was aspirated and 1.3 mL of prewarmed 39°C fixative (above) was applied.

Oocytes were fixed for 4 min at 39°C, washed briefly in 2xSSCT (Saline Sodium

Citrate + 0.1% Tween-20), and ovarioles separated by pipetting. Oocytes were

washed in 2xSSCT three times for 10 min, washed 10 min each in 2xSSCT

containing 20%, 40% and 50% formamide, then incubated in 2xSSCT + 50%

formamide for 2h at 37°C. As much buffer as possible was aspirated, and 40 µl of

hybridization solution (36 µl of 1.1x hybridization solution (1.0g dextran sulfate, 1.5

ml 20x SSC, 5.0 ml formamide, dilute to 9.0 ml with ddH2O) plus 4 µl of probe mix)

was added. All probes were synthesized with fluorophores by idtdna.com and diluted

to 200 ng/µl in ddH2O. Probe mixes were prepared by combining 2 µl of each probe

to be used then diluting to a total volume of 96 µl in ddH2O, then stored at -20°C.

Using 4 µl of probe mix applied 16.7 ng of each probe to the prep. Probes used were

2L-3L (AATAACATAG)3 and 4 (AATAT)6 (Dernburg, 2000) and X (TTT-TCC-AAA-

TTT-CGG-TCA-TCA-AAT-AAT-CAT) (Ferree and Barbash, 2009).

After the hybridization solution was added, DNA was denatured at 92°C

followed by overnight hybridization at 32°C. Oocytes were washed twice for 15 min

in 2x SSCT + 50% formamide at 32°C, for 10 min each in 2x SSCT containing 40%,

20%, and 0% formamide, then stained in 2x SSCT + 1x DAPI for 10 min. Oocytes

were washed in 2x SSCT (2x briefly, 2x 10 min), then mounted in SlowFade Gold.

Immunofluorescent Preps. 2 day mated females were dissected as per FISH

preps (1 min CO2, 5 min rest, quickly dissected then incubated for up to 10 min in

Robb’s), followed by fixation at room temperature in 1.3 mL fixative. Oocytes were

then washed briefly in PBST, ovarioles separated by pipetting, washed 3x for 10 min

in PBST. Oocytes were dechorionated by rolling between frosted glass slides,

washed 3x briefly in PBST, transferred to an 0.5 mL eppendorf tube and blocked for

1 hr in PBST-NGS (Matthies et al., 2000). Fresh PBST-NGS with primary antibodies

(Serotec MCA786 rat anti-tubulin at 1:250 and Millipore rabbit anti-phosphorylated-

histone H3 at serine 10 at 1:500) was added and hybridized overnight, followed by

washing in PBST (3x briefly, 1x 15 min), 1 hr blocking in PBST-NGS and then either

  18  

4 hr incubation at room temperature, or overnight at 4°C, in PBST-NGS plus

secondary antibodies (Goat anti-rat IgG with Alexa Fluor 647 conjugate and goat

anti-rabbit IgG with Alexa Fluor 568 conjugate, Invitrogen, both at 1:250). 2.5 µl of

200x DAPI was added and incubated for 6 min, followed by PBST washes (3x brief

and 2x 15 min) and mounting in SlowFade Gold.

Imaging and Quantification. To ensure oocytes were not missed or double

counted, microscope slides were photographed on a dissection microscope and a

print of the photo was used as a map to mark oocytes. Oocytes were viewed at low

magnification and marked using the LAS AF software (www.leica.com) Mark and

Find panel. All confocal images were collected with the 63x objective on a Leica

TCS SPE II confocal microscope using LAS AF and presented images were

deconvolved using Huygens Essential (www.svi.nl).

Estimation of 4-4 distances was done by combining XY distances (determined

by the LAS AF line tool in projected stacks) with Z distances (determined by

multiplying the number of confocal sections between the centers of the 4 light cones

by the section thickness in orthogonal projections) using the Pythagorean theorem

(distance = sqrt(xy2 + z2)) in Excel. Measurement was restricted to oocytes that had

at least one 4 out on the spindle. This was determined by whether there was at least

a 50% dip in background-subtracted fluorescent intensity, measured on the 4 and

the space between the 4 and the adjacent chromosome using the line ROI tool.

Oocytes with both 4s on the same side of the spindle, with additional nonexchange

chromosomes, or with chromosomes in the ‘slippage’ configuration (Hughes et al.,

2011) were counted as having chromosomes out on the spindle, but their 4-4

distances were not included in the analysis. Plots and t-tests were then done in R.

To calculate chromosome 4 brightness ratios, figures where both 4

chromosomes were fully separated from other chromosomes were selected,

identically-sized regions of interest (ROI) were placed over each 4 and on nearby

empty space, and the summed pixel intensity for each ROI was recorded. The

brightness ratio (lower intensity - background) / (higher intensity - background) was

calculated for 10 oocytes for each genotype.

  19  

Acknowledgments.

We thank Dr. J.P. Masly, Dr. Stuart MacDonald, and the Bloomington

Drosophila Stock Center (supported by NIH P40OD018537) for stocks, and Dr.

Giovanni Bosco, Dr. Keith Maggert, Dr. Sarah Zanders and Kevin Wei for helpful

comments. Supported by NIH GM074737 to D.A.B. and NIH GM099054 to W.D.G.

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Drosophila  melanogaster.  II.  Further  genetic  analysis  of  the  nod  locus.  Genetics  

125:  115-­‐127.

Zwick,  M.  E.,  J.  L.  Salstrom,  and  C.  H.  Langley,  1999  Genetic  variation  in  rates  of  

nondisjunction:  association  of  two  naturally  occurring  polymorphisms  in  the  

chromokinesin  nod  with  increased  rates  of  nondisjunction  in  Drosophila  

melanogaster.  Genetics  152:  1605-­‐1614.

  24  

Figure 1. Asymmetry in sim-IV heterozygotes. pol and ciD are visible

markers on different D. melanogaster chromosome 4s. Representative oocytes from

42-48 hour-old mated females from the DAPI-only preps used for 4-4 distance

measurement, scaled to the same size. The differences in the brightness of the 4s

are not as clear in these projected images as in the ocular, so the background-

subtracted intensity of each 4 was determined, and the brightness ratio (dimmer 4 /

brighter 4) calculated, for 10 oocytes per genotype, with the mean (and range)

reported. (A) A homozygous control pol/pol oocyte. Mean brightness ratio: 0.87

(0.77-0.98). (B) A heterozygous sim-IV/pol oocyte made from outcrossing the

introgression stock. The dimmer sim-IV chromosome is indicated (asterisk). Mean

brightness ratio: 0.63 (0.40-0.76). (C) A heterozygous sim-IV/ciD oocyte from the

introgression stock. The dimmer sim-IV chromosome is indicated (asterisk). Mean

brightness ratio: 0.66 (0.57-0.89). (D) A homozygous sim-IV/sim-IV oocyte from the

introgression stock. The 4s are dimmer but not asymmetric. Mean brightness ratio:

0.88 (0.73-0.96). (E) A pure-strain D. simulans oocyte. The 4s are also dimmer but

not asymmetric. Mean brightness ratio: 0.94 (0.78-0.99).

Figure 2. Heterochromatin threads in D. simulans. (A) A fixed oocyte from a

2-day-old mated D. simulans female, visualized by immunofluorescence with anti-

tubulin (red), anti-pH3S10 (white) and DAPI (blue) staining. Threads are detectable

by anti-pH3S10; the right chromosome has a clear and complete thread while a very

dim spur can be seen on the left chromosome (arrow). (B) A fixed oocyte from a 3-

day-old mated D. simulans female, visualized by heterochromatin FISH (white)

against the AATAT repeat primarily found on chromosome 4. A complete thread can

be detected running between 4 chromosomes.

Figure 3. 4-4 distance measurements. pol and ciD are visible markers on

different D. melanogaster chromosome 4s. (A) The mean distances for each

genotype (horizontal lines) and the inner quartile ranges (boxes) are indicated, along

with the number of measurements. The first four sets are for D. melanogaster,

including the pol/pol control, the outcrossed sim-IV/pol heterozygote, the

  25  

introgressed sim-IV/ciD heterozygote, and the introgressed sim-IV/sim-IV

homozygote, while the fifth set is for pure-strain D. simulans females. The sixth set is

D. melanogaster females heterozygous for the deletion Df(4)m101-62f/pol (see Fig.

4). Figure 4. Asymmetry in Df(4)m101-62f heterozygotes. A fixed oocyte from a

mated 2-day-old heterozygous Df(4)m101-62f/pol female is shown, with FISH

staining of the 359-bp satellite (X Probe, green), the AATAT repeat (4 Probe, red)

and the AATAACATAG repeat (2L3L Probe, white) along with DAPI (blue). The Df(4)

chromosome (asterisk) stains less brightly with both DAPI and the 4 probe,

consistent with the deletion of some AATAT heterochromatin from this chromosome.

Figure 5. Expected progeny from the cross in Table 1 to measure the sim-

IV segregation ratio. At top are two spindle diagrams, showing normal segregation

(left) and meiosis I nondisjunctional segregation (right). As either spindle pole can

form the egg pronucleus, those poles drop down to four types of female gametes in

the table. Chromosome loss is also possible but not diagrammed; in that case, nullo-

4 gametes equivalent to the last column will be produced. Females are mated to

compound-4 bearing males of genotype C(4), ci ey, who produce either diplo-4 or

nullo-4 gametes. Progeny will be y+ if the maternal y+-IV is transmitted, and are

otherwise y mutant, indicated by the background color. The hatching pattern

indicates progeny that are semi-viable or lethal. Haplo-4 leads to minute phenotypes

with poor viability, while nullo-4 is always lethal. Tetra-4 flies from nondisjunctional

oocytes are usually lethal, but can survive under some circumstances (Grell, 1972).

Note that the normal yellow+ triplo-4 progeny are indistinguishable from the

nondisjunctional diplo-4 progeny (as well as any tetra-4 progeny that survive).

Therefore only the yellow ci ey class of progeny from NDJ can be observed. A

similar situation arises in most of the crosses in Table 2, where sim-IV/pol progeny

arising from non-disjunction are phenotypically wild type and cannot be distinguished

from triplo-4 regular progeny. In both Tables 1 and 2, progeny inheriting no maternal

4 are products of either maternal non-disjunction or chromosome loss and are

  26  

detected by their ey ci phenotype. Although only half of the exceptional progeny are

therefore detectable, we have calculated 4 NDJ without doubling the number of

nullo-4 progeny observed, as spontaneous 4 NDJ events in wildtype and nod–

heterozygous backgrounds yielded 11 nullo events and only 1 diplo event across

multiple experimental controls (Gillies et al., 2013; Rasooly et al., 1991; Zhang and

Hawley, 1990), suggesting these arise primarily from loss events rather than

nondisjunction. Products of meiosis II non-disjunction are not shown, but again only

those inheriting no maternal 4 are phenotypically distinguishable.

Figure 6. Expected segregation types and phenotypic classes of progeny from triplo-4 females. The unmarked 4 being tested is indicated as “IV”. Triploid

females of chromosome 4 genotype y+-IV/w+-IV/IV were mated to y w/Y males with

unmarked 4s. Female chromosome 4s can segregate in three possible classes to

generate six different gametes. However not all gametes can be distinguished

because the tested 4 is unmarked, leading to the same phenotype from different

genotypes, as indicated by background colors. When the two marked 4s segregate

to opposite poles, the unmarked chromosome will segregate to either pole. This

leads to Class I segregations (y+-IV <=> w+-IV / IV) and Class III segregations (w+-IV

<=> y+-IV / IV), which both produce progeny carrying only one of the two 4-linked

markers. Conversely, in Class II segregations the two marked 4 chromosomes move

to the same pole, leading to progeny that are either wildtype or mutant for both

markers together. If segregation is equal, then all six classes of progeny are equally

likely, leading to an expected 2 : 2 : 1 : 1 ratio of the phenotypes y+ w : y w+ : y+ w+ :

y w.

  27  

Supporting Information

Figure S1 Significance of 4-4 distance measurements. The 4-4 distances for

each genotype in Fig. 3A were used to calculate all possible pairwise t-tests, with

genotypes in the same order as in Fig. 3A. The p values for each test are listed and

color coded according to the key. The bottom row shows the total number of oocytes

examined, along with the percentage of those oocytes with 1 or more chromosomes

out on the spindle (which indicates that the oocyte is in prometaphase) for each

genotype. Note that the number of oocytes out (“% out” times Total) is greater than

the N values listed in Fig. 3A, as oocytes with nonexchange chromosomes in

addition to IV or transient configurations such as slippage or with both homologs on

the same spindle arm were excluded from the 4-4 measurements, but were counted

here.

  28  

Table 1. Test of segregation, chromosome loss and NDJ Regular progeny Exceptional progeny

Chr. 4

tested

F1 Sex

No. inheriting

P[y+]

No. inheriting

tested

chromosome

Segregation

ratio a

No. 4 NDJ

4 NDJ % b

w+-IV Female 1249 1194 0.489 1

Male 1022 1095 0.517 1

Both 2271 2289 0.502 n.s. 2 0.044

sim-IV Female 1276 1147 0.473 0

Male 1031 963 0.483 1

Both 2307 2110 0.478 ** 1 0.023

y w; w+-IV females were crossed to w/Y; sim-IV/ciD males. y w/Y; w+-IV/sim-IV sons

were then crossed to y w; y+-IV females. y w; y+-IV/w+-IV and y w y+-IV/sim-IV

daughters were collected and separately crossed to y1 pn1/Y; C(4)RM, ci1 eyR/O

males at 27°. a Defined as the ratio of those inheriting the tested chromosome/total progeny. As

each class has a 50% chance of survival due to sperm genotype (Fig. 5),

significance was tested by comparison to simulation of equal segregation followed

by 50% survival with 1,000,000 replicates. n.s. = not significant (p > 0.5); ** = p<

0.002. b Calculated as the number of observed exceptional progeny/total progeny

(excluding minutes; see Fig. 5 and Methods). The NDJ rates for the two genotypes

were not significantly different (p = 1, Fisher’s Exact Test).

  29  

Table 2. Tests for sim-IV nondisjunction in multiple genetic backgrounds

Genotype Normal Progeny

4-only NDJ

X-only NDJ

X & 4 Double

NDJ

pol+ minute

a

pol– minute

a

X NDJ %

b

4 NDJ %

b y w; sim-IV/sim-IV 181 0 0 0 0 -- 0% 0% y w; sim-IV/ciD 230 0 0 0 2 -- 0% 0% y w; sim-IV/pol 1641 0 0 0 119 56 0% 0% y w/y w noda; pol 509 2 0 0 -- 235 0% 0.39% y w/y w noda; sim-IV/pol

866 4 1 0 133 135 0.23% 0.46%

FM7/y w; sim-IV/pol

1405 1 5 1 189 134 0.85% 0.21%

FM7/y w; sim-IV/sim-IV

1127 3 7 0 314 -- 1.22% 0.26%

Females of the indicated genotypes were crossed to C(1;Y), v f B/O; C(4)RM, ci eyR/O

males.

a The missing class of minutes cannot be produced by these crosses.

b The number of X NDJ progeny was doubled for calculation of X NDJ, to account for

inviable classes (Zeng et al., 2010). Number of X & 4 double NDJ progeny was

therefore also doubled for calculation of both X NDJ and 4 NDJ. In calculating

percentage of X NDJ and 4 NDJ the number of NDJ progeny was divided by the sum of

the total progeny, not including minutes.

  30  

Table 3. Triplo-4 segregation tests

Source of chr

4 tested

No. y+ No. w+ a No. y+ w+ No. y w b Class II freq. c

BS 1 725 714 285 307 29.1% ***

BOG 1 165 216 ** 151 133 42.7% ***

sim-IV 356 383 295 333 45.9% ***

VAG 1 151 167 136 171 * 49.1% ***

Wild 5B 141 131 140 131 49.9% ***

y w 670 760 * 741 901 *** 53.5% ***

y w; y+-IV / w+-IV / 4 females, where 4 represents the unmarked chromosome 4 being

tested, were crossed to y w/Y males. * = p<0.05; ** = p<0.01; *** = p<0.001 in chi-squared

tests.

a y+ and w+ classes were tested for deviation of a 1:1 ratio.

b y+ w+ and y w classes were tested for deviation of a 1:1 ratio.

c y+: w+: y+ w+: y w classes were tested for deviation from a 2:2:1:1 ratio.

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